Taq polymerase buffer

Variation in the ppAzoR gene was generated by using ep-PCR. Primers 5′-GGAGAGTCATATGAAACTGTTGC-3′ (PpaF) and 5′-CAACCAAAGGATCCCTTGATCAGG-3′ (PpaR) were used for amplification. Nucleotides for restriction sites of NdeI and BamHI are underlined. ep-PCR was carried out in 50 µL reaction volumes containing 3 ng of DNA template (plasmid pLP-1, where ppAzoR gene [29] (link) and variants are cloned), 0.5 µM of primers, 200 µM of dNTPs, 7 mM MgCl₂, Taq polymerase buffer, and 5 U of Taq polymerase (Fermentas). The effect of MnCl₂ was tested at 0.1–0.25 mM concentrations. After an initial denaturation period of 10 min at 94°C, the following steps were repeated for 30 cycles in a thermal cycler (MyCycler™ thermocycler, Biorad): 1 min at 94°C, 1 min at 55°C and 45 s at 72°C followed by a final 10 min period at 72°C. The amplified products were purified using GFX PCR DNA and Gel Band Purification kit (GE Healthcare). The final PCR products were digested with NdeI/BamHI (Fermentas) and cloned into pET-21a (+) (Novagen). Ligations were performed with T4 DNA ligase (Fermentas) using a 1∶8 vector to insert ratio. Reaction mixtures were incubated overnight at room temperature, incubated at 65°C for 10 min, and then used to transform electrocompetent E. coli KRX cells.

Thirty-three ISSR primers from primer set no. 9 (University of British Columbia, Canada) and 50 RAPD primers (Operon, Eurofins Genomics, India) were selected for this study based on the presence of clear, repeatable and polymorphic amplified bands. The amplification was carried out in 20 μL reaction volume and consisted of 0.1 mM of each dNTP, 1 U Taq polymerase, 1× of Taq polymerase buffer, 1.6 mM MgCl₂ (Fermentas, USA) and 20 ng genomic DNA. DNA amplification was performed in a thermocycler (Corbett Research, Australia) programmed for an initial denaturation at 94 °C for 5 min, 44 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C/37 °C (for ISSR and RAPD primers, respectively) for 45 s and extension at 72 °C for 1 min and a final extension at 72 °C for 10 min. The amplified products were separated on 2 % agarose gel and stained with ethidium bromide (2 μg mL⁻¹). The reproducibility of DNA amplification profiles was tested by repeating the polymerase chain reactions (PCRs) twice with 20 of the 33 selected ISSR primers and 20 of the 50 selected RAPD primers.

DNA from each sample was amplified by PCR with the primer sets described in Table 1. A 20 μL reaction was assembled that contained 100–300 ng sample DNA, 1x Taq polymerase buffer (Fermentas), 0.1 mM deoxynucleotide triphosphate (dNTPs), 2.5 mM MgCl₂, 1U of Taq polymerase (Fermentas), and 0.5 pmol of each primer. The PCR conditions for each primer set are detailed in Table 2. DNA from each sample was extracted in duplicate and each PCR reaction was performed in duplicate for each independent extraction to ensure consistency of the results. PCR reactions were carried out individually for each primer set and the bands produced in five reactions with each primer set were sequenced to ensure specificity of amplification.
The amplified products were analyzed on a 1.5% agarose gel, stained with ethidium bromide and visualized on a UV transilluminator. Beta-globin primers were used to check the quality of the DNA. The amplification was carried out in the presence of negative and positive controls; DNA from cervical cancer tissue samples positive for HPV (confirmed by sequencing) was used as a positive control for HPV16 and a negative control for HPV18-specific primer sets and DNA from HeLa cell line was used as positive control for HPV18 and a negative control for HPV16-specific primer sets.